XAFS studies of carboxypeptidase A: detection of a structural alteration in the zinc coordination sphere coupled to the catalytically important alkaline pKa.
XAFS studies of carboxypeptidase A: detection of a structural alteration in the zinc coordination sphere coupled to the catalytically important alkaline pKa.
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羧肽酶 A 的 XAFS 研究：检测锌配位层中与催化重要的碱性 pKa 相关的结构变化。
DOI：
10.1021/bi00213a013
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Auld,DS
中科院分区：
文献类型：
--
作者：
Zhang,K;Auld,DS
修订稿于 1993 年 9 月 28 日收到®摘要：羧肽酶 A (ZnCPD) 的 X 射线吸收精细结构 (XAFS) 光谱显示出渐进的光谱变化，特别是当 pH 从中性值变为碱性值时，在近边缘区域。最小二乘拟合和径向分布函数 (RDF) 数据分析均在所有 pH 值以及 150 和 297 K 下产生 ZnCPD 第一配位层中原子的两种分布。直接比较 ZnCPD 第一配位层和更高配位层揭示了 pH 7.0 和 pH 9.9 之间的结构差异。在 pH 7.0 时，锌离子具有 4 个配体原子（N 或 O），平均距离为 2.024±0.006 Á，并且在距锌离子 2.54±0.05 Á 处分布有 1.3 个原子（N 或 O）（来自最小二乘拟合分析）。在 pH 9.9 时，较大的分布在距锌 0.022 A 较短距离 (2.002 Á) 处包含 4 个原子，而较小的分布在 2.52±0.06 Á 处包含 0.7 个原子。较小的分布主要归因于 Glu 72 的 2-氧的贡献以及距离较远的原子的贡献，这些原子的贡献无法完全与第一个壳层峰分开。 ZnCPD 在中间 pH 值下的结构变化与在 pH 7.0 和 pH 9.9 下观察到的变化一致。 XAFS Debye-Waller 因子显示在碱性 pH 条件下四原子分布的结构无序性增加。较高壳层比较表明，两个组氨酸配体 His 69 和 His 196 在检查的 pH 值下保持不变。作为 pH 函数的吸收边标准化光谱变化图符合 pXa 9.49 at-4 C 的理论 pH 滴定曲线。该值对应于通过羧肽酶 A 催化三肽水解的动力学分析外推 pXeh 值而获得的值 (Auld & Vallee (1971) Biochemistry 10, 2892)。 pH 依赖性较短的平均金属配体距离和增加的第一壳结构无序最容易解释为其他两种金属配体之一（水分子或 Glu 72 的 «-氧）与金属在碱性 pH 值下移动 0.09±0.03 Á 更接近锌离子。这种情况可能是由于金属结合水的直接电离或 Glu 72 配位中电离相关的改变所致。如本文所讨论的，我们倾向于将动力学曲线 pXEH 中的碱性 p 分配给金属结合水分子的电离。
Revised Manuscript Received September 28, 1993® abstract: X-ray absorption fine structure (XAFS) spectra of carboxypeptidase A (ZnCPD) show progressive spectral changes particularly in the near edge region when the pH is changed from neutral to alkaline values. Both least square fitting and radial distribution function (RDF) dataanalysis yield two distributions of atoms in the first coordination shell of ZnCPD at all pH values and at both 150 and 297 K. Direct comparison of the first and higher coordination shells of ZnCPD reveals structural differencesbetween pH 7.0 and pH 9.9. At pH 7.0, the zinc ion has four ligand atoms (N or O) at an average distance of 2.024±0.006 Á, and a smaller distribution of 1.3 atoms (N or O) at 2.54±0.05 Á from the zinc ion (from the least square fitting analysis). At pH 9.9, the larger distribution contains four atoms at a 0.022 A shorter distance (2.002 Á) from the zinc, while the smaller distribution contains 0.7 atom at 2.52±0.06 Á. The smaller distribution can be attributed mainly to the contribution of the «2-oxygen of Glu 72 and to the atoms farther away for which the contribution cannot be fully separated from the first shell peak. The structural changes of ZnCPD at intermediate pHs are consistent with the changes observed at pH 7.0 and pH 9.9. The XAFS Debye-Waller factor shows an increased structural disorder for the four atom distribution at the alkaline pH. The higher shell comparison shows that the two histidine ligands His 69 and His 196 remain unchanged at the pHs examined. The plot of the normalized spectral changes at the absorption edge as a function of pH conformsto a theoretical pH-titration curve with a pXa 9.49 at-4 C. This value corresponds to that obtained by extrapolation of values of pXeh by kinetic analysis of the carboxypeptidase-A catalyzed hydrolysis of tripeptides (Auld & Vallee (1971) Biochemistry 10, 2892). The pH-dependent shorter average metal-ligand distance and the increased first shell structural disorder is most easily explained by one of the other two metal ligands (the water molecule or the «-oxygen of Glu 72) to the metal moving 0.09±0.03 Á closer to the zinc ion at alkaline pH values. Such a situation could be due to either direct ionization of the metal bound water or an ionization-linked alteration in the coordination of Glu 72. As discussed herein, we favor assigning the alkaline p in the kinetic profiles, pXEH, to the ionization of the metal bound water molecule.